BMP type I receptor inhibition reduces heterotopic [corrected] ossification

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Figure 1

Mouse model of FOP. (a) Staining of muscle sections from Ad.Cre-injected conditional caALK2–expressing (ALK^Q207D) mice. The injection done at P7 induced recombination in myocytes of the left (L) gastrocnemius and soleus muscles at P11, as evidenced by loss of nuclear β-galactosidase (β-gal) staining and gain of GFP expression. Squares indicate areas of low magnification examined at higher magnification to the right. H&E staining reveals mononuclear cell infiltrates and myocyte edema in tissues undergoing recombination, but not in uninjected right (R) hindlimb muscles. (b) Left hindlimb postural abnormalities were observed grossly (left) at P30 in the Ad.Cre-injected left hindlimbs of conditional caALK2 mice, but not in Ad.Cre-injected wild-type mice. The X-ray image (right) shows Ad.Cre-induced ectopic calcifications involving the left gastrocnemius, soleus and hamstring muscles of conditional caALK2 mice at P30. (c) Three-dimensional reconstructed images from μCT cross-sections of an Ad.Cre-injected, conditional caALK2–expressing mouse on P30 showing intramuscular ectopic bone within the left gastrocnemius, soleus, tibialis and hamstring muscles (rendered in light gray) fusing with the pelvis and proximal femur.

Figure 2

Effect of LDN-193189 on BMP signaling and function. (a) Structures of dorsomorphin and LDN-193189. (b) Quantitative immunoblotting of PASMCs showing differential effects of dorsomorphin or LDN-193189 on BMP4–induced (10 ng ml⁻¹) phosphorylation of Smad1, Smad5 and Smad8 (p-Smad1/5/8), with IC₅₀ values of ~400 nM and ~5 nM, respectively. (c) Immunoblot of PASMCs treated with LDN-193189 showing differential inhibition of BMP4 (10 ng ml⁻¹) or TGF-β (0.5 ng ml⁻¹) signaling (IC₅₀ ~ 5 nM and ≥ 1 μM, respectively). (d) Id1 promoter activity induced by transient transfection of COS cells with ALK2^R206H or ALK2^Q207D. Id1 promoter activity (BRE-Luc) was increased by 250- to 300-fold over control plasmid (pcDNA, n = 3 measurements, mean ± s.d., NS, no significant difference). (e) Impact of LDN-193189 on the transcriptional activities of caALK2^R206H and caALK2^Q207D mutants, expressed as percentage of full Id1 promoter activity (n = 3 measurements, mean ± s.d., *P < 0.05 versus untreated). (f) Impact of LDN-193189 (100 nM) on BMP4-induced (10 ng ml⁻¹) osteoblast differentiation of C2C12 cells at various intervals before and after BMP4 treatment (n = 6 measurements, mean ± s.d., *P < 0.01 versus BMP4 treatment alone) and on cell viability (bottom panel). (g) Top, immunoblot for p-Smad1/5/8 and total Smad1 in PASMCs expressing the conditional caALK2^Q207D transgene after infection with Ad.Cre or Ad.GFP showing the impact of pretreatment with LDN-193189 (100 nM). Bottom, immunoblot for phosphorylated Smad1/5/8 and total Smad1 in Ad.GFP- or Ad.Cre-infected PASMCs treated with BMP4 at varying concentrations.

Figure 3

Impact of LDN-193189 on ectopic ossification in vivo. (a) Conditional caALK2–expressing mice receiving vehicle after Ad.Cre injection on P7 developed radiographic disease at P13, progressing to fusion of left hindlimb joints by P30–P60, whereas treatment with LDN-193189 diminished ectopic bone formation and preserved joint spaces over the same interval without inducing fractures, osteopenia or skeletal abnormalities (n = 3–5 mice per treatment group; data are representative of six independent experiments). (b,c) Alizarin red and Alcian blue staining of mice at P15 showing ectopic calcifications encasing the left tibia and fibula in vehicle-treated conditional caALK2–expressing mice, but not in LDN-193189–treated mice. Higher magnification images are shown in c. Ectopic bone or cartilage are absent in the wild-type hindlimb. A-P, anterior-posterior; LAT, lateral; P-A, posterior-anterior. (d) μCT imaging showing attenuated ectopic calcification in LDN-193189–treated mice as compared to vehicle-treated mice at P15 and P30. (e) Fixed extension of left hip, knee and ankle joints, as evident in anesthetized and flaccid Ad.Cre-injected conditional caALK2–mutant mice at P30. The extension is attenuated in LDN-193189-treated mice. (f) Passive range of motion impairment score, as assessed by the minimum angle formed by the ankle and tibia with passive dorsoflexion. (g) Impact of vehicle and LDN-193189 treatment upon passive range of motion impairment in Ad.Cre-injected conditional caALK2–mutant mice at P15 and P30 (n as indicated, bars represent mean, *P < 0.001).

Figure 4

Impact of pharmacologic inhibition of BMP signaling and inflammation in the mouse FOP model. (a) Immunofluorescence showing enhanced nuclear p-Smad1/5/8 and expression of Runx2 in a number of recombined (β-gal–negative) myocytes from left (L) hindlimb muscles of Ad.Cre-injected, conditional caALK2–mutant mice at P11 (with DAPI counterstain in blue, right). Both are diminished in LDN-193189–treated mice. Uninjected right(R)-hindlimbs are shown as controls. (b) Histological evidence of intramuscular endochondral bone, as shown by alkaline phosphatase staining (AlkPhos) of osteoblasts, chondrocytes, matrix and marrow cells in recombined tissues of vehicle-treated, Ad.Cre-injected, caALK2-transgenic mice at P30. The staining is diminished and absent in LDN-193189–treated and uninfected mice, respectively (higher magnification, right panels). (c) High-frequency recombination evidenced by loss of β-gal staining in muscle, vascular and connective tissues of tamoxifen-treated, CAGGS-CreER:CAG-Z-EGFP-caALK2 mice but not single-transgenic CAG-Z-EGFP-caALK2 mice at P30. (d) Plain radiographs of tamoxifen-treated double- and single-transgenic mice at P60 show absence of ectopic calcification. (e) Alizarin red and Alcian blue staining revealing ectopic calcification in tamoxifen-treated, Ad.GFP-injected double-transgenic but not single-transgenic mice at P14. (f) Impact of dexamethasone treatment (10 mg kg⁻¹ daily) upon radiographic ossifications in Ad.Cre-injected, conditional caALK2–expressing mice at P15 and P30, as compared to vehicle treatment. (g) Impact of dexamethasone treatment upon impairment of passive range of motion (ankle flexion) in Ad.Cre-injected, conditional caALK2–expressing mice at P15 and P30 (data are representative of three independent experiments, n as indicated; values are mean ± s.d., *P < 0.05).